Side effects associated with opioid analgesics, such as nausea, drowsiness, respiratory depression, and potential for addiction, are motivating the design and development of new therapies for acute, subacute and chronic pain. Voltage-gated Na+ ion channels are integral membrane proteins responsible for the transmission of signals along electrically conducting cells. Ten mammalian genes have been sequenced, which encode ten distinct channel isoforms (NaV1.1-1.9 and NaX), each having unique gating properties, and cellular and tissue distribution patterns. Recent studies have correlated a hereditary loss-of-function mutation in one human Na+ channel isoform - NaV1.7 - with a rare genetic disorder known as Congenital Insensitivity to Pain (CIP). Individuals with CIP have reduced sensitivity to normally painful stimuli without significant deficits to sensory or cognitive function. A compellin body of evidence indicates that selective inhibition of NaV1.7 in normal humans could recapitulate the phenotype of CIP. The high homology of human NaV proteins, coupled with challenges associated with high-throughput screening against multiple ion channel targets, have thwarted most efforts to develop selective antagonists for individual NaV subtypes. Recent findings indicate that a two amino acid variation in the pore region of hNaV1.7 is responsible for reduced potency of a family of naturally-occurring sodium channel antagonists, the guanidinium toxins (GTxs), against this isoform. This variation is present in all known hNaV1.7 splice variants, but is not found in any other human NaV isoform. In Phase I of our SBIR program, GTx analogues were designed and synthesized that exhibit selective inhibition of hNaV1.7 over other NaV isoforms as measured by whole-cell patch clamp electrophysiology. In Phase 2 of our program, we aim to improve the selectivity and drug properties of our lead compounds with a focused, medium-throughput medicinal chemistry effort, and to evaluate the most promising candidates as therapeutics for pain in preclinical safety and efficacy studies. Success of this program will lead to the nomination of one or more high-affinity, isoform-selective inhibitors of NaV1.7 as drug candidates for pain treatment.